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Fc blocked

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Fc-blocked is a product designed for cell-based assays. It functions to reduce non-specific binding of antibodies to Fc receptors, which can interfere with experimental results.

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Lab products found in correlation

Ionomycin, by BioLegend (2 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions) Phorbol myristate acetate (pma), by BioLegend (2 mentions) Rhodium cell id, by Standard BioTools (2 mentions) Cytofix cytoperm kit, by BD (2 mentions) Brefeldin a, by BioLegend (2 mentions) Cd19 pecy5, by Thermo Fisher Scientific (2 mentions) Anti mouse siglecf pe, by BD (1 mentions) Cd11b percp, by BioLegend (1 mentions) Tcrβ pacific blue, by BioLegend (1 mentions) Ly6c apc cy7, by BD (1 mentions) Cd11c apc, by Thermo Fisher Scientific (1 mentions) Ly6g pe cy7, by BioLegend (1 mentions) Lsrii fortessa flow cytometer, by BD (1 mentions) Igd pe, by Thermo Fisher Scientific (1 mentions) Cd8 fitc, by Thermo Fisher Scientific (1 mentions) Cd4 apc, by Thermo Fisher Scientific (1 mentions) Cd3 apc, by Thermo Fisher Scientific (1 mentions) Cd45ro pe, by Thermo Fisher Scientific (1 mentions) Cd27 fitc, by Thermo Fisher Scientific (1 mentions)

7 protocols using fc blocked

1

Cytokine Stimulation and CyTOF Profiling

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Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
Ho W.J., Zhu Q., Durham J., Popovic A., Xavier S., Leatherman J., Mohan A., Mo G., Zhang S., Gross N., Charmsaz S., Lin D., Quong D., Wilt B., Kamel I.R., Weiss M., Philosophe B., Burkhart R., Burns W.R., Shubert C., Ejaz A., He J., Deshpande A., Danilova L., Stein-O’Brien G., Sugar E.A., Laheru D.A., Anders R.A., Fertig E.J., Jaffee E.M, & Yarchoan M. (2021). Neoadjuvant cabozantinib and nivolumab convert locally advanced hepatocellular carcinoma into resectable disease with enhanced antitumor immunity. Nature cancer, 2(9), 891-903.
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2

Cytokine Stimulation and CyTOF Profiling

Cited 1 time
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Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
Ho W.J., Zhu Q., Durham J., Popovic A., Xavier S., Leatherman J., Mohan A., Mo G., Zhang S., Gross N., Charmsaz S., Lin D., Quong D., Wilt B., Kamel I.R., Weiss M., Philosophe B., Burkhart R., Burns W.R., Shubert C., Ejaz A., He J., Deshpande A., Danilova L., Stein-O’Brien G., Sugar E.A., Laheru D.A., Anders R.A., Fertig E.J., Jaffee E.M, & Yarchoan M. (2021). Neoadjuvant cabozantinib and nivolumab convert locally advanced hepatocellular carcinoma into resectable disease with enhanced antitumor immunity. Nature cancer, 2(9), 891-903.
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3

Immune Cell Profiling in Murine Sepsis

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LPS (6 mg/ml) and/or AD-01 (3 mg/kg) or equal volume sterile PBS was injected via intraperitoneal injection into C57BL/J6 wild-typeor Fkbpl +/-mice. Following a 3 h incubation mice were culled by cervical dislocation and peritoneal lavage was performed with 3 ml of PBS containing 3% BSA. Lavage fluid was harvested and cells were Fc blocked (Invitrogen, Ireland) and stained on ice for 30 min (Table 3) and gated as outlined in Supplementary Figure 2.
Compensation was determined by single staining alone and no positive staining detected from IgG controls. Analysis was conducted using FlowJo Version 2 (Flowjo, USA).
Annett S., Spence S., Garciarena C.D., Campbell C., Dennehy M., Drakeford C., Lai J., Dowling J.K., Moore G., Yakkundi A., Short A., Sharpe D., Furlong F., O’Donnell J.S., Cavalleri G.L., Kerrigan S.W., Tikhonova I.G., Johnson P., Kissenpfennig A., & Robson T. (2021). The immunophilin protein FKBPL and its peptide derivatives are novel regulators of vascular integrity and inflammation via NF-κB signaling.
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4

Characterization of Airway Immune Cells

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Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).
dos-Santos J.S., Firmino-Cruz L., da Fonseca-Martins A.M., Oliveira-Maciel D., Perez G.G., Roncaglia-Pereira V.A., Dumard C.H., Guedes-da-Silva F.H., Santos A.C., Leandro M.D., Ferreira J.R., Guimarães-Pinto K., Conde L., Rodrigues D.A., Silva M.V., Alvim R.G., Lima T.M., Marsili F.F., Abreu D.P., Ferreira Jr. O.C., Mohana Borges R.D., Tanuri A., Souza T.M., Rossi-Bergmann B., Vale A.M., Silva J.L., de Oliveira A.C., Filardy A.D., Gomes A.M, & de Matos Guedes H.L. (2022). Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum. Frontiers in Immunology, 13, 884760.
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5

Comprehensive Immune Cell Profiling

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Single cell suspensions were washed with PBS and 1.0 × 106 cells/tube were FC blocked (eBioscience) and stained extracellularly for 30 min at 4 °C. Cells were then washed and prepared for intracellular staining using the FoxP3 Intracellular Staining Kit (R and D systems). Intracellular staining was performed at 4 °C for 60 min. Antibodies CD79A-APC, CD27-FITC, IgD-PE, CD4-APC, CD45RO-PE, CD8-FITC, and CD3-APC were purchased from eBioscience.
Centuori S.M., Gomes C.J., Kim S.S., Putnam C.W., Larsen B.T., Garland L.L., Mount D.W, & Martinez J.D. (2018). Double-negative (CD27−IgD−) B cells are expanded in NSCLC and inversely correlate with affinity-matured B cell populations. Journal of Translational Medicine, 16, 30.
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6

Adoptive Transfer of OT-1 CD8+ T cells

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OT-1 CD8+ T cells (in C57BL/6 background) were isolated from the spleens of CD45.2 mice and 1 × 106 cells were transplanted into C57BL/6 CD45.1 hosts. 24 h later, transplanted and control mice were injected i.p. with 100 μg OVA, 100 μg anti-CD40, and 50 μg Poly (I:C). Peripheral blood was acquired through submandibular bleed every 7 days thereafter. Peripheral blood was RBC lysed (ACK Buffer, Fisher) and Fc-blocked (eBioscience) and WBCs were stained with CD45.1, CD45.2, and CD8 (eBioscience) and KB-OVA tetramer (MLB International) in PBS supplemented with 0.5% Bovine Serum Albumin for 30 min on ice. Cells were washed twice and data was collected on an LSRII (Beckman Dickinson) and analyzed with FlowJo 9.6 (TreeStar). Cells were gated on strict forward and side scatter parameters, to ensure single cell analysis. Additionally, the DAPI- population was used for viable cells. Finally, cells were separated by CD45.1 or CD45.2 positivity, and assessed for CD8/OVA-tetramer expression.
Bronk C.C., Yoder S., Hopewell E.L., Yang S., Celis E., Yu X.Z, & Beg A.A. (2014). NF-κB is crucial in proximal T-cell signaling for calcium influx and NFAT activation. European journal of immunology, 44(12), 3741-3746.
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7

BAL Immune Cell Profiling by Flow Cytometry

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Flow cytometry was used to determine the number and types of cells present in the BAL fluid. Fc-blocked (1 µg/ml; eBiosciences) BAL cells were stained with anti-mouse SiglecF-phycoerythrin (PE; 1 µg/ml; BD Pharmingen), CD45- allophycocyanin (APC; 1 µg/ml; eBiosciences), CD3-PECy5 (1 µg/ml; eBiosciences), CD19-PECy5 (1 µg/ml; eBiosciences), CD11c-PECy7 (1 µg/ml; eBiosciences), MHCII-APC-efluor780 (1 µg/ml; eBiosciences), CD11b-V450 (1 µg/ml; BD Pharmingen), Ly6C-FITC 1 µg/ml; BD Pharmingen) and Ly6G-AlexaFluor700 (1 µg/ml; BD Pharmingen). Fixable Viability Dye eFluor® 506 (eBiosciences) was added to exclude dead cells from the analysis.
Joe P.T., Christopoulou I., van Hoecke L., Schepens B., Ysenbaert T., Heirman C., Thielemans K., Saelens X, & Aerts J.L. (2019). Intranodal administration of mRNA encoding nucleoprotein provides cross-strain immunity against influenza in mice. Journal of Translational Medicine, 17, 242.
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